A New Methodology for Assessing Macromolecular Click Reactions and its Application to Amine-tertiary Isocyanate Coupling for Polymer Ligation Guillaume Gody, 1,* Derrick A. Roberts, 2 Thomas Maschmeyer 3 and Sébastien Perrier 1 * 1 Department of Chemistry, The University of Warwick, CV4 7AL, UK. 2 Department of Chemistry, The University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom 3 Laboratory of Advanced Catalysis for Sustainability, School of Chemistry, The University of Sydney, NSW 2006, Australia * Correspondence to Tel: +44 (0)2476 528085; Fax: +44 (0)2476 524112; E-mail: s.perrier@warwick.ac.uk; g.gody@warwick.ac.uk EXPERIMENTAL PARTS MATERIALS AND METHODS Materials. 1,4-Dioxane (Sigma-Aldrich, 99%), tetrahydrofuran (THF, Ajax Finechem, 99%), N,N-dimethylformamide (DMF; Merck, HPLC-grade, 99%), n-hexane (n-hex; Redox Chemicals, 99+%), ethyl acetate (EtOAc; Ajax Finechem, 99%), dichloromethane (CH 2 Cl 2 ; Ajax Finechem, 99.7%), diethyl ether (Et 2 O; Merk, 99%), toluene (Merk, 99.8%), methanol (MeOH; Redox Chemicals, 99+%), dimethyl sulfoxide (DMSO; Sigma-Aldrich, 99%), triethylamine (Et 3 N; Ajax Finechem, 99%), diphenyl phosphoryl azide (DPPA; Sigma Aldrich, 97%), N-methyl-morpholine (NMM; Sigma-Aldrich, 99%), benzylamine (BzNH 2 ; Sigma-Aldrich, 99%), benzylmercaptan (BzSH; Sigma-Aldrich, 99%), tert-butyl isocyanate (tbunco; Sigma-Aldrich, 97%), ethylenediamine (H 2 N-CH 2 -CH 2 -NH 2 ; Sigma-Aldrich, 99%), dibutyltin dilaurate (DBTDL; Sigma-Aldrich, 95%), 1,2-ethanedithiol (HS-CH 2 -CH 2 - SH; Sigma-Aldrich, 98%), ethylene glycol (HO-CH 2 -CH 2 -OH; Sigma-Aldrich, 99%), 1,8- diazabicycloundec-7-ene (DBU; Sigma-Aldrich, 98%), 1-butanethiol (Sigma-Aldrich, 99%), sodium hydroxide (NaOH; Sigma-Aldrich, 97%, pellets), carbon disulfide (CS 2 ; Sigma- Aldrich, 99%, anhydrous), 2-bromo-2-methylpropionic acid (Sigma-Aldrich, 98%), N- hydroxysuccinimide (NHS, Sigma-Aldrich, 98%), 4-(dimethylamino)pyridine (DMAP; Sigma-Aldrich, 99%), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), iodine (I 2 ; Sigma-Aldrich, 99%) were used without further purification. 4-Acryloylmorpholine (NAM, Sigma-Aldrich, 97%), tert-butyl acrylate (tba; Sigma-Aldrich, 98%) and methyl methacrylate (MMA; Sigma-Aldrich, 99%) were filtered through basic aluminium oxide (activated, basic, Brockmann I, standard grade, ~150 mesh, 58 Å) before use to remove the radical inhibitor. 2,2ʹ-Azobis(2-methylpropionitrile) (AIBN) was purified by recrystallization from MeOH. Piperazine hexahydrate (Sigma-Aldrich) was dried in a desiccator with phosphorus pentoxide. Milli-Q water was obtained from a Millipore Milli-Q Plus water purification system. All polymerizations were carried out under an argon atmosphere. Silica gel (Grace, 63 μm) was used to perform column chromatography. The collected fractions were analyzed by thin layer chromatography (TLC-plates, Merk 60 F254). S1
Methods Nuclear Magnetic Resonance (NMR) spectroscopy ( 1 H NMR). Spectra were recorded on a Bruker Avance DPX 300 spectrometer (300 MHz) at 27 C either in deuterated chloroform (CDCl 3 ) or in deuterated dimethylsulfoxide (DMSO-d 6 ). Chemical shift values (δ) are reported in ppm, using the residual solvent signals as an internal reference. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR). Spectra were recorded using a Bruker Alpha-E FTIR spectrometer fitted with a zinc-selenide crystal in the region between 4000 and 400 cm -1. The resolution was set-up at 4 cm -1, the scan speed at 0.5 cm s -1 with 120 scans performed per sample. Size Exclusion Chromatography (SEC). Number-average molar masses (M n,sec ) and dispersity values (Ð) were determined using size exclusion chromatography either with THF or DMF as an eluent. The THF system was equipped with a Jordi Gel DVB 500 Ǻ guard column (50 7.8 mm) followed by two PLgel MIXED-C columns (300 7.5 mm). The flow rate of the eluent (THF containing 0.04 g L 1 hydroquinone) was set to 1.0 ml.min 1 at 40 C. Polystyrene standards were used to calibrate the SEC system. Analyte samples contained toluene (0.5 vol. %) as the flow rate marker and were filtered through polytetrafluoroethylene (PTFE) membranes (with 0.45 μm pore size) before injection (100 μl). Experimental molar masses were determined by conventional calibration using ASTRA for Windows software. The DMF system was fitted with a PolarGel-M guard column (50 7.5 mm) and two PolarGel-M analytical columns (300 7.5 mm). For this system, the eluent (DMF + 0.04 g.l 1 hydroquinone + 0.1 wt. % lithium bromide) flow rate was set at 0.7 ml min 1 and the temperature was set to 50 C. Poly(methyl methacrylate) standards were used as calibrants. Analyte samples contained 0.5 vol. % water as the flow rate marker and were filtered through PTFE membranes with 0.45 μm pore before injection (100 μl). Molar masses were obtained by conventional calibration using Cirrus software. Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). Measurements were performed with a micro-tof (Bruker Daltonics) mass spectrometer equipped with an automatic syringe pump for sample injection. The mass spectrometer was operating in the positive ion mode. The standard electrospray ion source was used to generate the ions. The ESI micro-tof MS instrument was calibrated in the m/z range from 100 to 3000 g mol 1 using an internal calibration standard (Tunemix solution). Data were processed via Bruker Data Analysis software version 4.0. Determination of monomer conversions. Monomer conversions (p) were calculated from 1 H NMR data using the following equation: p = 1 (( I 5.5-6.75 ppm / 3) / DP targeted ), with I 5.5-6.75 ppm the integrals of the three vinyl protons from the monomer, I a the integral of the 3 methyl protons calibrated at 3 belonging to the Z group of the RAFT agent (-CH 2 -CH 3 ), and DP targeted the average degree of polymerization targeted. S2
Calculation of M n,th. The theoretical number-average molar masses (M n,th ) were calculated using Equation S1 neglecting the term 2f[I] 0 (1-e -kdt )(1-f c /2). [M] 0 pm M [M] 0 pm M M n,th = + M CTA + M CTA (S1) k t f d c [CTA] [CTA] 0 0 + 2 f [I] 0(1 e )(1 ) 2 where [M] 0, [CTA] 0, [I] 0 are the initial concentrations (in mol.l 1 ) of the monomer, the chain transfer agent and the initiator, respectively; p is the monomer conversion as determined by 1 H NMR; M M and M CTA are the molar masses (in g.mol 1 ) of the monomer and the chain transfer agent, respectively; k d is the decomposition rate constant (in s 1 ) of the azoinitiator; t represents the polymerization time (in seconds). The factor 2 in Equation 1 accounts for the fact that one molecule of azoinitiator yields two primary radicals with the efficiency f. The term 1-(f c /2) represents the number of chains produced in a radical radical termination event with f c representing the coupling factor. Theoretical estimation of the percentage of α-end functional polymer chains. The theoretical percentage of polymer chains bearing a functional group in the α-chain end (i.e., derived from the R group of the RAFT agent) was estimated using Equation S2. [CTA] 0 α = (S2) k t f d c [CTA] 0 + 2 f [I] 0(1 e )(1 ) 2 RAFT polymerization. CTA, monomer and azoinitiator are charged into a flask having a magnetic stir bar. The flask is sealed with a rubber septum and bubbled with argon for 10 minutes. The solution is then allowed to stir at the desired temperature in a thermostated oil bath for the desired time. After reaction, the mixture is cooled in an ice bath to room temperature and open under air. A sample is taken for 1 H NMR (to determine monomer conversion) and SEC analysis (to determine M n.sec and Ð). For one-pot post-polymerization reaction, 0.1 to 0.5 equivalent of the linker (ethylenediamine or ethylene glycol or 1,2-ethane dithiol or diamino cyclic peptide) is directly added to the polymerization mixture at room temperature under air. Sample analysis by ESI TOF MS or by SEC is performed directly on the reaction mixture without purification. S3
Estimation of the coupling efficiency from SEC data. To determine the efficiency in polymerpolymer coupling, the SEC distribution w(log M) vs Log M is first converted into number distribution P(M) vs M according the method of Zetterlund et al. 1 where the y-axis, given in SEC, is defined as follow: - in SEC distribution: y-values are proportional to nm 2 (where M and n denote MW and the number of molecules); - in number distribution: y-values are proportional to n It follows that the number distribution, where the y-values are proportional to the number of chains, is given by w(log M)/M 2. By plotting w(log M)/M 2 vs M, the area under the curve between two given values of M is proportional to the number of chains within that range of M values. Quantification of the coupling efficiency (i.e., coupled vs. non-coupled polymer chains) is then determined by multiple peak-fitting analysis of the SEC traces plotted in terms of number distribution (P(M) vs M). Multiple peak-fitting was performed using the Multipeak Fiting 2 data analysis package contained within Wavemetrics IgorPro 6.34A. Each trace was automatically fitted with a linear baseline correction function and multiple Gaussian components until a small and randomly varying residual trace was obtained. Figure S1 shows a representative example of the peak-fitting procedure for a single SEC trace. Residual 1.0 0.5 0.0-0.5-1.0 Chain Number Distribution 8 6 4 2 0 8 6 4 2 0 5 0 1 10 Molecular Weight (kda) Figure S1. Representative example of the Gaussian multiple peak-fitting procedure used to analyse SEC traces for determining polymer polymer coupling efficiency. Baseline, peak identification and fitting were performed using the Multi-peak Fiting 2 data analysis package included in IgorPro 6.34A. 15 20 S4
RAFT agent synthesis. Synthesis of 2-(((butylthio)carbonothioyl)thio)-2-methylpropanoic acid To a solution of butanethiol (5 ml, 45.90 mmol) in acetone (2.5 ml) at room temperature under air was added a 20 wt% aqueous solution of sodium hydroxide (9.15 g, containing 1.83 g, 45.9 mmol of NaOH). The clear, colorless solution was stirred for 30 min. then treated with carbon disulfide (3.10 ml, 51.6 mmol) to give an orange solution. After further 30 min stirring, the reaction mixture was cooled in an ice bath and 2-methyl 2-bromopropanoic acid (8 g, 0.005 mol) was added slowly such that the temperature did not exceed 30 C. Then a 20 wt % aqueous solution of sodium hydroxide (9.40 g, containing 1.88 g, 47.0 mmol of NaOH) was added at such a rate that the temperature did not exceed 30 C. When the exotherm had ceased, the ice bath was removed, and the reaction was stirred for 24 h. The reaction mixture was diluted with water (50 ml), cooled in an ice bath (2-3 C) and HCl was added slowly until the CTA precipitated (ph ~2-3). The RAFT agent was recovered by vacuum filtration, washed with cold water, then dissolved in dichloromethane and dried over magnesium sulfate. After filtration and removal of the dichloromethane under reduce pressure, the powdery yellow solid is dry on vacuum (8.34 g, 33.05 mmol, 72 % yield) (see Figure S2a for the 1 H NMR spectrum). 1 H-NMR (300 MHz, CDCl 3, ppm): δ = 3.29 (2H, t, -CH 2 -S), 1.80-1.55 (8H, m, (CH 3 ) 2 -C and -CH 2 ), 1.42 (2H, m, -CH 2 ), 0.93 (3H, t, J = 7 Hz, -CH 3 ). Synthesis of 1-azido-2-methyl-1-oxopropan-2-yl butyl carbonotrithioate (CTA A) To a solution of 2-(((butylthio)carbonothioyl)thio)-2-methylpropanoic acid (1.5 g, 5.9 mmol) in dichloromethane (4 ml) at room temperature under air was added triethylamine (920 µl, 6.54 mmol), followed by a solution of diphenylphosphoryl azide (2.0 g, 7.1 mmol) in dichloromethane (5 ml) dropwise over 15 minutes. After addition, the mixture was stirred for 2 h at room temperature (see Figure S2b for the 1 H NMR spectrum after 2 h). After evaporation of the solvent under reduced pressure at 20 C, the mixture was purified by silica gel column chromatography (eluent: n-hexane/et 2 O, 98/2 v/v) to afford the desired product (1.15 g, 4.15 mmol, 70 % yield) as an orange oil (see Figure S2c for the 1 H NMR spectrum). S5
1 H-NMR (300 MHz, CDCl 3, ppm): δ = 3.30 (2H, t, J = 7.4 Hz, -CH 2 -S), 1.75-1.65 (8H, m, (CH 3 ) 2 -C and -CH 2 ), 1.44 (2H, m, -CH 2 ), 0.93 (3H, t, J = 7.5 Hz, -CH 3 ). Figure S2: 1 H NMR spectra (300 MHz, CDCl 3 ) of (a) the starting CTA, 2- (((butylthio)carbonothioyl)thio)-2-methylpropanoic acid, and (b) the reaction mixture after 2 h and (c) the CTA 1-azido-2-methyl-1-oxopropan-2-yl butyl carbonotrithioate (CTA A) obtained after purification by silica gel column chromatography. S6
Synthesis of 2,5-dioxopyrrolidin-1-yl 2-(((butylthio)carbonothioyl)thio)-2-methylpropanoate (CTA B) To a solution of 2-(((butylthio)carbonothioyl)thio)-2-methylpropanoic acid (1.00 g, 3.96 mmol) in dichloromethane (20 ml) at room temperature under air was added N- hydroxysuccinimide (558 mg, 4.75 mmol) and 4-Dimethylaminopyridine (DMAP) (63.6 mg, 0.515 mmol). Then a solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (0.939 g, 4.75 mmol) in dichloromethane (10 ml) was added dropwise over a period of 20 min at room temperature. The reaction was allowed to proceed at room temperature for a further 16 h, yielding an orange solution. Excess EDC and DMAP were removed by washing with water (2 80 ml) and once with brine (80 ml). The CH 2 Cl 2 phase was dried over MgSO 4, filtered and evaporated under reduce pressure. Silica gel column chromatography was performed using hexane:ethyl acetate (70:30 v/v) as the eluent. Purified CTA B was isolated as an orange oil that solidified in the fridge (1.17 g, 3.37 mmol, 85% yield) (see Figure S3 for the 1 H NMR spectrum). 1 H-NMR (300 MHz, CDCl 3, ppm): δ = 3.32 (t, 2H, S- CH 2 -), 2.81 (s, 4H, -CH 2 -CH 2 -, N-hydroxysuccinimide), 1.88 (s, 6H, -C(CH 3 ) 2 -S-), 1.68 (quintet, 2H, S-CH 2 -CH 2 -), 1.43 (quintet, 2H, -CH 2 -CH 3 ), 0.93 (t, 3H, -CH 3 ). Figure S3: 1 H NMR spectrum (300 MHz, CDCl 3 ) of CTA B obtained after purification by silica gel column chromatography. S7
Figure S4. Kinetics of the benzylamine reaction with tert-butyl isocyanate performed in an NMR tube at 25 C ([Bz-NH 2 ] 0 = ([tert-butyl-nco] 0 = 30 mm). The amine-isocyanate reaction is very fast and efficient, with near quantitative coupling obtained in c.a. 1 h. S8
Figure S5. ESI-MS spectra of the polymer Bz-PNAM 10 in presence of benzylamine (2 equiv.) obtained after (a) 2 h reaction time and (b) 24 h reaction time. Incipient aminolysis is apparent after 2 h, indicated by the mixture of Bz-PNAM 10 -TTC and Bz-PNAM 10 -SH, and proceeds to completion after 24 h. S9
Figure S6. ESI-MS spectrum of NCO-PNAM 10. Inset: HRMS analysis of [(NCO- PNAM 6 )+Na] + molecular ion. Figure S7. ESI-MS spectrum of Bz-PNAM 10 in the presence of benzylamine (1 equiv.) obtained after 2 h reaction time and (b) 24 h reaction time HRMS analysis of [(Bz- PNAM 6 )+Na] + molecular ion. S10
Figure S8. HRMS analysis of [(Bz-PNAM 6 -SH)+Na] + molecular ion. Figure S9. Chromatograms (in SEC-THF) showing the coupling reaction (after 1 hour) between the α-nco-pmma 30 and ethylenediamine (0.5 equiv.) performed at 25 C in nondry dioxane directly after polymerization (one-pot reaction, run 4, 93±1.0% coupling efficiency). S11
Figure S10. Chromatograms (in SEC-THF) showing the coupling reaction between the α- NCO-PtBA 33 and (a) ethylenediamine (0.5 equiv.) (one-pot reaction, run 3, 96 ± 1.2% coupling efficiency) or (b) piperazine (0.5 equiv.) (one-pot reaction, run 4, 95 ± 1.7% coupling efficiency) performed at 25 C in non-dry toluene directly after polymerization. S12
Figure S11. Chromatograms (in SEC-THF) showing the coupling reaction (after 1 h) between (a) the α-nco-pnam 100 and ethylenediamine (0.5 equiv.) (one-pot reaction, run 5, 91 ± 1.1% coupling efficiency) and (b) the α-nco-pnam 200 and ethylenediamine (0.5 equiv.) (one-pot reaction, run 6, 78 ± 1 % coupling efficiency) both performed at 25 C in non-dry dioxane directly after polymerization. S13
Scheme S1. One-pot synthesis of polymer-cyclic peptide conjugates via amine t NCO coupling (run 9, Table 1). α-nco-pnam 40 was synthesized from CTA A. No purification of the polymer was required since full monomer conversion is achieved. The diamino-cyclic peptide (0.5 equiv.) was added to the polymerization mixture as a solution in non-dry DMSO. Coupling was allowed to proceed at room temperature (~25 C) for 4 days to 1 week. S14
Scheme S2. One-pot synthesis of polymer-cyclic peptide conjugates via amine t NHS active ester coupling (run 10, Table 1). α-nhs-pnam 40 was synthesized from CTA B. No purification of the polymer was required since full monomer conversion is achieved. The diamino-cyclic peptide (0.5 equiv.) was added to the polymerization mixture as a solution in non-dry DMSO. Coupling was allowed to proceed at room temperature (~25 C) for 4 days to 1 week S15
Figure S12. In situ 1 H NMR study of the reaction velocity for the reaction of benzylmercaptan (1 equiv.) with tert-butyl isocyanate (1 equiv.) (a) Timecourse 1 H NMR spectra (300 MHz, 298 K, CDCl 3 ) (b) plot of the consumption of the isocyanate against reaction time. S16
Figure S13. Chromatograms (SEC-THF) showing the coupling reaction between α-nco- PNAM 40 and 1,2-ethanedithiol (0.5 equiv) performed directly after polymerization at 25 C in non-dry dioxane in the presence of (a) Et 3 N (1 equiv.) (run 11, Table 2) and (b) DBU (1 equiv.) (run 12, Table 2). S17
Figure S14. Chromatograms (SEC-THF) showing the coupling reaction (after 48 hours) between the α-nco-ptba 39 and ethylene glycol (0.5 equiv) performed directly after polymerization at 25 C in non-dry toluene in presence of DBTL as a catalyst (run 13, Table 2, 51 ± 2.0% coupling yield). S18
Figure S15. Chromatograms (SEC-THF) showing (a) the RAFT polymerization of NAM with (full line) or without (dashed line) 10% of water and (b) the coupling efficiency of the α- NCO-PNAM 40 obtained in RAFT polymerization with 10% water with ethylenediamine (run 16). The shoulder at low molar mass most likely represents the polymer chains that have coupled only once with ethylene diamine, which was in excess due to the NCO degradation. S19
Figure S16. (a) Comparison of the SEC chromatograms before and after precipitation in a mixture of MeOH/H 2 O 50/50 v/v at 0 C showing the stability of the isocyanate group (run 17, Table 3); (b) FT-IR spectra showing the perfect retention of the NCO group; (c) the coupling reaction (full line) between ethylenediamine and α-nco-pba 35 (dash line) obtained after precipitation. The approximation in the stoichiometry might explain the shoulder observed at low molar mass. S20
Table S1: Experimental conditions used for the preparation of various α-functional polymers with the CTA A (carbonyl azide) or B (NHS active ester) following by the one-pot addition of a linker (ethylene diamine or piperazine or diamino-cyclic peptide). Run 1 2 3 4 5 CTA A A A A A monomer NAM MMA tba tba NAM m CTA (mg) 29.3 34.6 49.2 48.2 12.2 m monomer (g) 0.6 0.5 0.900 0.89 0.626 m AIBN solution [a] (g) 0.179 0.178 [b] 0.253 0.248 0.097 m solvent (g) 0.732 0.149 0.074 0.075 0.819 V total (ml) 1.417 0.908 1.404 1.389 1.444 Stock solution of diamine (mg ml -1 ) 15.9 4.7 5.3 7 4.7 V solution (ml) 0.200 0.798 1.003 1.068 0.056 [diamine] 0 (mm) 33 37 36.8 35 13 Run 6 7 8 9 10 CTA A A B A B monomer NAM NAM NAM NAM NAM m CTA (mg) 9.8 14.5 37.1 3.9 4.9 m monomer (g) 1 0.3 0.6 0.08 0.08 m AIBN solution [a] (g) 0.120 0.089 0.180 0.060 [c] 0.060 [c] m solvent (g) 1.398 0.367 0.731 0.024 0.030 V total (ml) 2.361 0.708 1.417 0.283 0.283 Stock solution of diamine (mg ml -1 ) 4.7 12.6 12.8 15 15 V solution (ml) 0.226 0.025 ( 5) 0.050 ( 5) 0.618 0.618 [diamine] 0 (mm) 7 31 30 8 8 a AIBN solution at 5 mg ml -1 ; b AIBN solution at 10 mg ml -1 ; c AIBN solution at 2 mg ml -1 S21
Table S2: Experimental conditions used for the preparation of various α-nco-polymers with the CTA A (carbonyl azide) following by the one-pot addition of a linker (1,2-ethanedithiol or ethyleneglycol). Run 11 12 13 CTA A A A monomer NAM NAM tba m CTA (mg) 29.4 29.4 32.5 m monomer (g) 0.6 0.6 0.6 m AIBN solution [a] (g) 0.180 0.180 0.192 d m solvent (g) 0.731 0.731 0.051 V total (ml) 1.417 1.417 0.936 Stock solution of 1,2-ethanedithiol (mg ml -1 ) 20 22.2 - Stock solution of ethylene glycol (mg ml -1 ) - - 18.5 V stock solution linker (ml) 0.050 ( 5) 0.250 0.200 V sol. Et3N (ml) 0.249 b - - V sol. DBU (ml) - 0.323 c - V sol. DBTDL (ml) - - 0.400 e [diamine] 0 (mm) 28 27 39 a AIBN solution at 5 mg ml -1 ; b Et 3 N solution at 21.5 mg ml -1 in dioxane; c DBU solution at 10 mg ml -1 in dioxane; d AIBN solution at 10 mg ml -1 ; e DBTDL solution at 36.6 mg ml -1 ; Table S3: Experimental conditions used for the preparation of α-nco-polymer and reaction with ethylenediamine in presence or not of water. Run 14 15 16 17 CTA NCO-PNAM 40 NCO-PNAM 40 A* A* monomer - - NAM BA m CTA (mg) - - 19.6 23.9 m monomer (g) - - 0.4 1.4 m AIBN solution [a] (g) - - 0.120 0.390 m solvent (g) - - 0.426 (diox) 0.059 (H 2O) 0.148 V total (ml) - - 0.944 2.185 Mass of polymer (g) 0.5 0.5-0.5 g Stock solution of ethylenediamine (mg ml -1 ) - 5 [b] 2.6 [c] 1.2 [d] V stock solution of ethylenediamine (ml) - 0.635 0.835 2.63 V H2O (ml) 1.9 1.5 - - [diamine] 0 (mm) - 25 20 20 * CTA A = isobutyryl azide butyl trithiocarbonate a AIBN solution at 5 mg ml -1 ; b ethylenediamine solution in H 2 O; c ethylenediamine solution in dioxane; d reaction performed on 0.5 g of PBA 35. (1) Zetterlund, P. B.; Kagawa, Y.; Okubo, M. Chemical Reviews 2008, 108, 3747. S22